1. Field of the Invention
This invention relates generally to biosensors; and more particularly to a biosensor method for detecting nitrate based on the fluorescence properties of a receptor molecule and a biosensor apparatus containing the active-site fragment of the receptor molecule for detecting nitrate.
2. Related Art
Nitrate ion from fertilizers and treated sewage has reached disquietingly high concentrations in water supplies all around the world. In the United States, the Environmental Protection Agency (EPA) has fixed an allowable upper limit of 10 ppm for NO3− nitrogen (NO3—N) in drinking water. This is to prevent illnesses caused by higher nitrate levels such as methemoglobinemia (“blue baby syndrome”) in bottle-fed infants. The health and environmental risks associated with elevated nitrate levels are the following:                Methemoglobinemia. Elevated nitrate levels poses a risk to infants and can lead to methemoglobinemia, or “blue baby syndrome”. Elevated levels of nitrate lead to a build-up of nitrite in the gastrointestinal tract by nitrate reducing bacteria. The excess nitrite moves into the bloodstream where it binds strongly to blood hemoglobin and impairs the delivery of oxygen to the baby.        A recent study from the University of Iowa has shown a link between nitrate levels in drinking water and bladder cancer in women (Weyer, et al., 2001)        Blood and serum nitrate levels can become elevated as the result of increased production of nitric oxide (NO). Nitric oxide is an unstable gaseous compound that readily diffuses into body fluids where it can be converted to nitrate, nitrite or S-nitrothiol. NO levels rise during heightened immune-response such as occurs during sepsis, organ failure or graft-rejection.        There is also a concern over excess nitrates and aquatic biology. When a nitrogen limited eco-system is supplied with high levels of nitrate, significant increases in the levels of phytoplankton (algae) and macrophytes (aquatic plants) can occur. This poses a significant threat to these fragile ecosystems. The recommended levels of nitrates to avoid the propagation of algal blooms is between 0.1 to 1 mg/l (NOAA/EPA).        
As the major environmental release of nitrate arises from its use in fertilizers, it is unlikely that the nitrate problem will disappear anytime soon. Thus, there will be a continued need to monitor nitrates in finished drinking water, watersheds, industrial wastewater, private wells and estuaries. Additionally, nitrate contamination of source water will always be a concern for industries that depend on water purity for the manufacturing of their finished product. The data related to nitrate as a contaminant demonstrates the scope of the problem:                According to the Toxic Release Inventory database nearly 60 million pounds of nitrate were released into water between 1987 and 1993. An additional 53 million pounds of nitrate was released into land over this same period. Nitrate is highly soluble and only weakly retained by soils, such that a large portion of the nitrate released to the ground will eventually end up in the water.        According to the EPA there were 14,000 measurement/recording violations for nitrate in the fiscal year 2000. These involved over 11,000 systems and affected over 4 million citizens. Similar numbers were recorded for the years between 1997 and 1999.        According to the EPA statistics regarding nitrate violations, for the fiscal year 2000, there were 804 violations occurring in 457 sites affecting a population of approximately 460,000 people with nitrate levels that exceeded the maximum contaminant level (MCL).        The number stated above for nitrate violations does not reflect the additional potential for exposure to elevated nitrate levels in the more than 15 million private wells in the United States. A 1992 survey conducted by the Office of Pesticides and Toxic substances of the EPA, estimated that 22,000 infants less than one year of age had well-water that exceeded the 10 ppm standard.        
It is therefore imperative to develop a reliable, sensitive and selective device to monitor drinking water for nitrate ions. There are a number of commercially available kits for measuring nitrate. These kits utilize a variety of sensing technologies. The EPA Office of Ground Water and Drinking Water maintains a database of approved analytical methods for drinking water compliance monitoring. The methods currently approved for monitoring nitrates are cadmium reduction, ion chromatography and ion-specific electrodes. It is our belief that none of these approaches provides a measurement technology that is rugged, sensitive and suited to the broad spectrum of water sources that need to be monitored. The most sensitive devices, such as ion chromatography, are not portable or adaptable for field-testing without shipping the samples. While many of the field test kits are portable they introduce the opportunity for operator error, in terms of mixing the reagents and interpreting the results. A survey of current nitrate detection technologies is presented in FIG. 22.
The Safe Drinking Water Act (SDWA) is the main federal law that ensures the quality of Americans' drinking water. Under the SDWA the United States Environmental Protection Agency (USEPA) has established guidelines and standards for drinking water quality. In 1996, Congress amended the SDWA to emphasize the importance of sound scientific assessment of the health risks related to water pollutants and contaminants. Our drinking water has shown remarkable improvements since the SDWA was adopted, however, there are growing concerns about the future of safe drinking water and water resources in the United States.                The cost associated with ensuring the safety of our drinking water is growing and will require a considerable input to upgrade the deteriorating water infrastructure in the United States.        Rural and tribal populations in the United States that do not have water that meets current standards. The prospect of increasing cost is an even greater concern to the rural water community, where economics of maintaining a safe water supply are the greatest challenge.        The standards may not be sufficient to ensure the safety of certain vulnerable sub-populations such as the elderly, infants, pregnant women and the immuno-compromised. A University of Iowa study has shown that the incidence of bladder cancer was nearly 3 fold higher for the group of women whose water supply had an average nitrate level of 2.46 mg/L nitrate-nitrogen versus those whose water supply contained an average of 0.36 mg/L nitrate-nitrogen. Alarmingly, this level is below the standard indicated under the SWDA.        There is a heightened concern about the health risks associated with exposure to contaminants such as arsenic, nitrate, heavy metals, disinfection by-products and other agents via drinking water. There is the prospect that even in the face of increasing operational cost to produce safe water that we may need to regulate and monitor even more contaminants.        
Effective monitoring is a critical component for providing clean, safe drinking water and protecting our water resources. The technology associated with water monitoring must be upgraded to meet the needs of the water and wastewater industries.                There is a need to increase our overall monitoring capabilities to accurately assess the effectiveness of government sponsored water resource management programs.        To develop cost-effective monitoring and processing technologies that will allow the rural and tribal communities to attain a high standard for their drinking water without taxing their limited economic resources.        To produce devices that emphasize simplicity and multi-contaminant analytical capabilities that will enable the individual operator to work more efficiently. This will eliminate the need for third party testing and concerns over shipping and custody of samples.        Design devices for in-line monitoring with direct data read-out to provide operators with critical information in real-time. This is essential for prompt decision making during critical events such as spills and floods.        
The quantitative assessment of single common or trace amounts of nitrate in solution depends primarily on chemical and/or analytical separation and detection technologies. These methodologies often require sample preparation, the use of various reagents and some physical transducer of the final product of the chemistry to provide quantitative information. Examples of physical transducers include optical detection, electrochemical, and a broad number of physical detection modes. See Riedel, K., 1998, in Ramsay, G. [ed.] Commercial Biosensors, Vol. 148, Chemical Analysis, John Wiley & Sons, New York, pp. 267–294; Kress-Rogers, E. 1997, Handbook of Biosensors and Electronic Noses: Medicine, Food, and the Environment, [ed., Kress-Rogers, E.], CRC Press, Boca Raton. In general, these technologies are time consuming, costly and require skilled operators but can provide sensitive and reliable quantification of specific analytes. Analytical methods that are rapid and perhaps less costly, may not be as sensitive or reliable as transducer methods, but may still meet the detection and/or quantification requirements.
More recently, sensors based upon biological sensing elements have been developed and exploited for detecting and quantifying a broad range of analytes from ions, metals, and small organics to proteins, lipids, nucleic acids and even whole organisms. These elements include enzymes, antibodies, RNA/DNA probes, membrane channels, whole cells, organs and even whole multicellular organisms. These types of sensors are called biosensors in that the sensing element is of biological origin.
Biosensors are monitoring devices composed of two elements, the first of which is the signal capture component that uses a biological entity such as an enzyme, antibody or cell surface receptor. The second part is the signal transduction element that converts the biological response into a measurable signal like fluorescence, electric current or potential. Biosensors have been described for the determination of more than thirty different environmentally relevant compounds (Riedel, 1998).
Biosensors can achieve the same or greater selectivity and sensitivity as analytical methods, and many allow detection and/or quantification in the absence of reagents and sample preparation, and most often do not require a skilled operator. Because the sensing element in a biosensor is typically very small and because detection is based upon molecular recognition of individual ligand molecules, biosensor devices can be very small and portable, thereby greatly expanding the utility and application of sensing and monitoring technologies.
Biosensors for a broad range of analytes including environmental contaminants and analytes relevant to industrial processes, medical diagnostics and law enforcement have been reported in the scientific and patent literature, though only a few technologies have obtained commercial success to date. See Riedel, K., 1998, in Ramsay, G. [ed.] Commercial Biosensors, Vol. 148, Chemical Analysis, John Wiley & Sons, New York, pp. 267–294; Kress-Rogers, E. 1997, Handbook of Biosensors and Electronic Noses: Medicine, Food, and the Environment, [ed., Kress-Rogers, E.], CRC Press, Boca Raton; Scheller, F. W. and Pfeiffer, D. 1997, in id; Urban, G. 1997 in id.
Enzyme-based biosensors that exploit oxidoreductases have been described. Nitrate reductase (NR), an oxidoreductase, from a variety of sources (bacteria, fungi, and vascular plants) has been used to assay nitrate in environmental or medical samples, in biosensor applications and in bioremediation applications. Nitrate reductases (NR) from different eukaryotic genera (yeast, algae, vascular plants) all share a common subunit structure and a catalytic function—the reduction of nitrate (NO3−) to nitrite (NO2−). A number of amperometric sensors exploiting various nitrate reductases have been described. As in any amperometric sensor, the Faradic current derived from the redox reaction at the electrode is measured. Glazier, S. A., Campbell, E. R. and Campbell, W. H. (1998, Anal. Chem. 70:1511–1515) generated an NR-based nitrate sensor that exploits a vascular plant (corn) NR and glassy carbon electrodes for the measurement of nitrate in buffered solutions.
Amperometric biosensors have been developed to take advantage of the redox properties of enzymes. In some applications, the enzymes may be maintained in solution on the surface of the electrode by using a semi permeable membrane, or they may be immobilized onto the surface of the electrode either covalently through some cross-linking chemistry or entrapped in a cross-linked matrix which adheres to the surface of the electrode. In the latter case, the matrix may be a protein or sol-gel, while in other it may be a conducting polymer that can serve to provide and enhance the electrical continuum between the redox centers of the enzyme and the electrode.
Moretto et al. (1998, Anal. Chem. 70:2163–2166; Ramsay, G. and Wolpert, S. M. 1997, Polymeric Mat. Sci. Engineer. 76:612–613) used an ultrathin film composite membrane technology to generate a nitrate biosensor. An ultra thin film of 1-methyl-3-(pyrrol-1-methyl) pyridinium tetrafluorborate was polymerized on an alumina support membrane, which has been coated with a film of gold. This film blocked the loss of methyl viologen, the electron donor to NR, and the free solution of Aspergillis sp. NR while allowing anions (e.g., nitrate) to flow freely to the enzyme. The enzyme activity was coupled to a glassy carbon electrode for amperometric assessment of nitrate levels in buffered solutions and in buffered natural water samples. In all cases where NR was “wired” with alkylpyrroleviologen-based redox polymers, enzyme activity was low. More recently, it has been demonstrated that such redox polymers and even the monomers in solution strongly (>90% loss of activity) inactivate NR (Ramsay and Wolpert, 1999, Anal. Chem. 71:504–506
Essentially all enzyme-based NR biosensors described to date lack stability, ruggedness or real-world applicability. In general, they show very limited periods of operational activity, from a few hours to a couple of days even under laboratory conditions. Lack of long-term stability and functionality typically has been ascribed to enzyme instability, loss of required enzyme mediators or both. Though numerous attempts have been made to overcome these features that limit their practical utilization and commercialization, we believe the present invention overcomes the bulk of the shortcomings of the existing technologies.
Enzyme-based amperometric sensors generally suffer from several major limitations: 1) traditional methods of electrode preparation with each of the three electrode cells comprised of different materials make modeled performances difficult to derive, 2) insufficient enzyme availability/high cost of enzyme preparation, 3) instability of enzyme and/or mediators under ambient conditions, 4) inadequate transducers for reporting enzyme activity, 5) inefficient enzyme immobilization or coupling to electrode, 6) end-product inhibition, and 7) enzyme specificity lacking, 8) a high cost of production and/or multiple steps in preparation.
As can now be seen, the related art remains subject to significant problems, and the efforts outlined above—although praiseworthy—have left room for considerable refinement.